NL2022942B1 - Use of iron chelate to reduce gastrointestinal tract colonisation by campylobacter - Google Patents

Abstract

The present application provides methods for preventing or reducing the prevalence of Campylobacter in a non-human animal host by administering iron(lll) chelate to said animal. The application further provides lron(lll) EDTA for use in the treatment of a Campylobacterinfection in an animal suffering from symptoms of said infection.

Description

USE OF IRON CHELATE TO REDUCE GASTROINTESTINAL TRACT COLONISATION BY CAMPYLOBACTER

FIELD OF THE INVENTION The present invention relates to a uses and methods of preventing or reducing the colonization of the gastrointestinal tract of an animal or human subject with Campylobacter.

BACKGROUND OF THE INVENTION Campylobacter, and particularly Campylobacter jejuni, is the most common cause of food poisoning in humans. Campylobacter is present in the gastrointestinal tract of animals, especially chicken, and may be transferred via their feces to food, such as raw or under- cooked meat, or drinking water. Once consumed, the bacteria adhere to the intestinal epithelium or mucus layer, causing toxin-mediated inhibition of fluid reabsorption from the intestine and invasion-induced inflammation and diarrhea. Symptoms of food poisoning from Campylobacter, including diarrhea, dizziness, stomach pain, muscle pain, headache, and fever, usually occur 2 to 5 days after a person eats contaminated food or drinks contaminated water. In exceptional cases, Campylobacter may cause the Guillain-Barré syndrome. Campylobacter infection may be diagnosed by conducting stool analysis. The stool of an infected person contains this bacterium in a large number. Hence, this condition can be identified by culturing and examining the stool under a microscope.

In young animals, such as calfs, Campylobacter jejuni can also cause gastrointestinal campylobacteriosis which is characterized by diarrhea. Campylobacter fetus can cause venereal disease and abortion in cattle.

Campylobacter jejuni infection is not currently considered to be pathogenic in poultry, but they tend to multiply in large numbers in the hindgut, principally in the caecae. From an economic perspective, it is common practice to overstock a shed with birds and thin some out after a few weeks. Campylobacter is often spread from flock to flock at this “thinning” stage. Densely housed birds kept in low light tend to panic and defecate when catchers enter sheds, causing further contamination. In order to control the rate of Campylobacter infections, in some countries, the caecae of a small selection of birds are sampled at the slaughterhouse and tested for the presence of Campylobacter. The farmer then typically receives a penalty for positive testing at the slaughterhouse.

Treatment of Campylobacter infections, currently involves the use of antibiotics, such as fluoroquinolones. However, mis-use and over-use of antibiotics has favoured the development of Campylobacter strains with reduced susceptibility to one or more of these antibiotics.

Therefore, a need exists for treatments of gastrointestinal infections in animals which have similar effects as antibiotic treatments but do not entail the risk of resistance, that are environmentally-friendly and which are less expensive than existing treatments.

Furthermore, strategies to prevent and reduce the microbial colonization in animals for human consumption, such as poultry, become more critical. One or more of these needs are met by the treatment of the present invention.

SUMMARY OF THE INVENTION The application provides methods for preventing or reducing the prevalence of Campylobacter in a non-human animal host, said method comprising administering iron(lll) chelate to said animal.

In particular embodiments of the methods provided herein, iron (lll) chelate is iron(lll) EDTA, DTPA, HEDTA, MGDA or GLDA. Most preferably the iron (lll) chelate is iron(lIHEDTA.

In particular embodiments of the methods provided herein, said animal is a live-stock animal and said iron (lll) chelate is administered to said animal prior to slaughter of said non-human animal.

In particular embodiments of the methods provided herein, the animal is a live-stock and the iron (lll) chelate is administered to said animal prior to envisaged events which increase the risk of infection or cross-infection of said animal.

In particular embodiments of the methods provided herein, the iron(lll) chelate is administered in the animal drinking water.

In particular embodiments of the methods provided herein, the animal is provided with drinking water comprising at least 5ppm iron(lll) chelate ad libidum.

In particular embodiments of the methods provided herein, the Campylobacter is Campylobacter Jejuni or Campylobacter Coli. The application also provides Iron (lll) chelates, such as Iron{(lIEDTA for use in the treatment of a Campylobacter infection in an animal suffering from symptoms of said infection.

The application also provides the use of iron(lll} chelate for preventing the invasion of gastrointestinal cells of an animal by Campylobacter or reducing the number of Campylobacter invading the gastrointestinal cells of an animal. In particular embodiments the use comprises administering iron(lll) EDTA in an effective amount to said animal to prevent the invasion of gastrointestinal cells of an animal by

Campylobacter or to reduce the number of Campylobacter invading the gastrointestinal cells of the animal The application also provides the use of iron (lll) chelate for preventing or reducing the transcellular migration of Campylobacter through the gastrointestinal cells of an animal. In particular embodiments, the use comprises administering iron(lll) EDTA in an effective amount to said animal to prevent or reduce the transcellular migration of Campylobacter through the gastrointestinal cells of the animal. The application also provides the use of iron (Ill) chelate for reducing the number of Campylobacter present in the gastrointestinal tract of an animal. In particular embodiments, the use comprises administering iron(lIINEDTA in an effective amount to said animal to reduce the number of Campylobacter present in the gastrointestinal tract of said animal. The application also provides the use of iron (lll) chelate for preventing or reducing the colonization of the gastrointestinal tract of an animal with Campylobacter. In particular embodiments, the use comprises administering iron(lII}EDTA in an effective amount to said animal to prevent or reduce the colonization of the gastrointestinal tract of said animal. By preventing the Campylobacter to invade the cells of the gastro-intestinal tract, they will not proliferate and thus not be able to create/continue a biofilm. The application thus also provides the use of iron (lll) chelate for preventing or reducing biofilm formation of Campylobacter in the gastrointestinal tract of an animal. In particular embodiments, said use comprises the administration of the iron(lll) EDTA to said animals in an effective amount to prevent said Campylobacter from forming a biofilm in the gastrointestinal tract of said animal or to reduce the amount of biofilm formed by Campylobacter in the intestinal tract of said animal. The application also provides the use of iron (lll) chelate for the prevention or reduction of transmission of Campylobacter infection from one animal to another.

BRIEF DESCRIPTION OF DRAWINGS Figure 1. Table 2. Cell adherence and invasion of Campylobacter jejuni 33291 in the presence of the 5 different iron chelate/zinc chelate formulations. QC: no treatment; A: 100% iron chelate/0% zinc chelate; B: 80% iron chelate/20% zinc chelate; C: 50% iron chelate/50% zinc chelate; D: 0% iron chelate/100% zinc chelate; E: 0% iron chelate/0% zinc chelate. Figure 2. Table 3. Cell adherence and invasion of Campylobacter field strains in the presence of the 5 different iron chelate/zinc chelate formulations. QC: no treatment A:

100% iron chelate/0% zinc chelate; B: 80% iron chelate/20% zinc chelate; C: 50% iron chelate/50% zinc chelate; D: 0% iron chelate/100% zinc chelate; E: 0% iron chelate/0% zinc chelate. Figure 3. Table 4. Effect of lron(lll} chelate on bird performance. Figure 4. Reduction of Campylobacter infection pressure in broiler flocks upon treatment with chelated iron in three farms

DETAILED DESCRIPTION OF THE INVENTION The term “about”, when used in relation to a numerical value, has the meaning generally understood in the relevant art. In certain embodiments the term “about” may be left out or may be interpreted to mean the numerical value +10%; or +5%; or +2%; or +1%. Whenever used herein in relation to a percentage, w/w means weight/weight and w/v means weight/volume. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms “comprising”, “comprises” and “comprised of’ when referring to recited members, elements or method steps also include embodiments which “consist of” said recited members, elements or method steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements or steps and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment envisaged herein. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments.

Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the 5 following claims, any of the claimed embodiments can be used in any combination.

Any references cited herein are hereby incorporated by reference.

The term “livestock” as used herein refers to domesticated animals raised in an agricultural setting to produce labor and commodities such as meat, eggs, milk, fur, leather, and wool.

The term “host” as used herein when referring to an infection by bacteria refers to a non-human animal which carries the bacteria without showing any clinical symptoms. Reference to “clinical symptoms” of an infection in an animal as used herein refers to features linked to the infection which are experienced as unpleasant and prevent the normal functioning of said animal.

The present invention is based on the observation that iron chelate, and particularly iron(lll) EDTA or an alkali salt thereof, can be advantageously used to reduce the incidence and severity of gastrointestinal infections caused by Campylobacter. It is envisaged that the invention is of use both in the context of general disease management and in the specific treatment of affected individuals.

More particularly, the inventors have surprisingly found that the administration of iron chelate to an animal, such as in the drinking water of said animal, prevents or reduces the number of Campylobacter in the gastrointestinal tract of the animal, reducing or preventing colonization of the gastrointestinal tract by Campylobacter and/or reducting or preventing transmission of Campylobacter between animals. Additionally, the biofilm formation by Campylobacter may also be reduced or prevented. The inventors found that these effects are mainly achieved by preventing or reducing invasion of and/or transcellular migration through the gastrointestinal cells of said animals by Campylobacter . Furthermore, these effects are also the result of a reduced adhesion of Campylobacter to the gastrointestinal tract of said animals. The reduction of the prevalence or the prevention of the presence of Campylobacter in an animal, such as in livestock or house animals is of interest to reduce the incidence of infection in consumers and/or keepers of these animals. In addition, the reduction or absence of Campylobacter may also have beneficial effects for the animal host, such as improved growth, increased weight, reduced susceptibility to other infections etc.

In particular embodiments, iron chelate is used for treating non-human animals for human consumption, as the fewer Campylobacter that are present in the gastrointestinal tract of such a non-human animal at the time of slaughter, the lower the risk of contamination of meat from the non-human animal with Campylobacter.

Furthermore, the fewer Campylobacter that are in the gastrointestinal tract of an animal, the lower the chance that the Campylobacter will spread from one animal to another.

As a result hereof, human subjects are less likely to consume contaminated food (e.g. contaminated raw meat of animals infected with Campylobacter) or contaminated water and are accordingly less likely to suffer from Campylobacter gastrointestinalinfections and the symptoms (e.g. diarrhoea) resulting from such infections.

The iron chelate further also allows to treat an animal infected with Campylobacter.

For example, administration of iron chelate to a human subject suffering from Campylobacter infection decreases or even complete eliminates the symptoms typically associated with Campylobacter infection, such as diarrhea.

In particular embodiments, iron chelate is thus used for treating a Campylobacter infection, in an animal showing symptoms (e.g. diarrhoea) resulting from such infection.

Present inventors have found that administration of iron chelate, and particularly iron(lll) EDTA, to an animal advantageously leads to a reduction of the number of Campylobacter that invade into and/or transmigrate through gastrointestinal cells, resulting in the prevention or reduction of damage to cell integrity of the intestinal cells caused by the pathogenic challenge.

Accordingly, one aspect provided herein is the use of iron chelate, preferably iron(lll) EDTA, for preventing the invasion of and/or transmigration through gastrointestinal tract of an animal by Campylobacter or reducing the number of Campylobacter invading and/or transmigrating through the gastrointestinal tract of an animal comprising administering iron(lll) EDTA in an effective amount to said animal to prevent the invasion of and/or transmigration through gastrointestinal tract of an animal by Campylobacter or to reduce the number of Campylobacter invading and/or transmigrating through the gastrointestinal tract of the animal.

In particular embodiments the iron(lll) chelate is used as a disinfectant of the gastro- intestinal tract, avoiding potential pathogenic effects of Campylobacter in the animal as well as preventing infection in humans ingesting the food products derived from the animal.

Accordingly, also provided herein is iron chelate, preferably iron(lll) EDTA, for use in the prevention or treatment of an infection by Campylobacter.

Also provided herein is a method for preventing the invasion of and/or transmigration through gastrointestinal tract of an animal by Campylobacter or reducing the number of Campylobacter invading and/or transmigrating through the gastrointestinal tract of an animal, the method comprising the step of administering iron chelate, preferably iron(lll) EDTA, in an effective amount to said animal to prevent the invasion of and/or transmigration through gastrointestinal tract of the animal by Campylobacter or reduce the number of Campylobacter invading and/or transmigrating through the gastrointestinal tract of the animal.

In particular embodiments, the number of Campylobacter invading and/or transmigrating through the gastrointestinal tract of the animal may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or at least 99%, preferably at least 50%.

In particular embodiments, the number of Campylobacter invading and/or transmigrating through to the gastrointestinal tract of the animal are the number of Campylobacter invading and/or transmigrating through to the large intestine of the animal.

In particular embodiments, the number of Campylobacter invading and/or transmigrating through to the gastrointestinal tract of the animal are the number of Campylobacter invading and/or transmigrating through to the gastrointestinal cells, such as the epithelial cells of the large intestine of the animal.

The term “invade” as used herein refers to the infiltration, entering into or penetration of the cell wall of intestinal cells.

The iron chelate of the uses and methods as taught herein, is preferably a chelate of iron, wherein said iron has 3 positive charges, i.e. Fe(3+), Fe(lll), Fet+++, Fe3+, Fer(3+), iron(lll), or iron(3+) and a molecular weight of 55.843 g/mol. Suitable chelating agents or agents for forming iron chelates are known in the art and include ethylenediaminetetraacetic acid (EDTA), pentetic acid or diethylene triamine pentaacetic acid (DTPA), 2.2-dipyridylamine (HDPA), N-{hydroxyathyil giulame aoid-N Nediacetic sold (GLDA) and the like. In particular embodiments, the iron chelate is selected from iron EDTA, iron DTPA, and iron HDPA. The skilied person will understand that iron ions may form charged complexes with the {conjugate bases of) the chelating agents, and that the complexes may be provided with counterions such as alkalimetal ions to obtain neutral chelates.

The present inventors have observed optimal effects with iron(lll) EDTA. Furthermore, iron chelate and especially iron(lll) EDTA is cheaper and easier to produce than known compounds, such as ferric quinate, used to reduce the incidence and severity of gastrointestinal infections caused by Campylobacter. Accordingly, the present invention allows reducing the costs associated with the prevention or reduction of transmission of Campylobacter infection from one non-human animal to another, meat production, disinfection of food stuff, and/or treatment of Campylobacter infections In particular embodiments, the iron chelate is an EDTA chelate of iron, preferably an EDTA chelate of iron(lll), in particular the salts of EDTA chelates of iron. EDTA chelates are widely available and are well tolerated. Of interest are the alkalimetal salts of iron[EDTA]! . Of particular interest are the salts of the iron chelates, such as Fe(lINK-EDTA or Fe(lll) potassium salt, or other salts such as sodium or ammonium salt. Preferably these salts are soluble in water. The potassium salts of iron[EDTA]" are attractive due to their solubility and effectiveness, thereby reducing amount of iron to be ingested, and reducing concomitant side effects in animals.

In particular embodiments, the iron chelate is iron(lll} EDTA or an alkali salt thereof, such as iron(lll) potassium EDTA.

The term “iron(lll} EDTA”, “(mono)ferric EDTA” or “Fe(lll) EDTA” as used herein, refers to ferric ethylenediaminetetraacetic acid (EDTA) with CAS number 15275-07-7, molecular formula C19H:2FeN:2Os and molecular weight: 344.06 g/mol.

The term “animal” as used herein may denote any type of animal, including humans, but also encompasses non-human animals that will be slaughtered for human consumption, such as cattle, sheep, ostriches, pigs, goats, deer, fish, shellfish and poultry (including birds such as chickens, geese, turkeys and ducks) or animals held as pets, such as dogs and cats and small farm animals. As chickens are a leading source of human infection with Campylobacter, the non-human animals are preferably poultry, more preferably chickens. In particular embodiments, the non-human animal are animals that can be treated through their drinking water, such as pigs and ostridges. It is clear to the skilled person that if the use or method involves slaughtering the animals, non-human animals are intended.

In particular embodiments, the animal is an animal comprising a detectable number of Campylobacter in its gastrointestinal tract. The presence of Campylobacter in the gastrointestinal tract may be determined methods known by the skilled person for identifying the presence of bacteria in the gastrointestinal tract. For example,

demonstrating the presence of the bacteria in the gastrointestinal tract or in a fecal sample of the animal by special culture techniques and molecular technologies (e.g. ELISA, PCR), depending on the suspected bacteria. In particular embodiments, the uses and methods as taught herein may comprise identifying the bacterial infection prior to starting the administration of the iron chelate as taught herein.

In particular embodiments, the animal is an animal showing or susceptible of showing one or more clinical symptoms of a Campylobacter infection, such as diarrhea, dizziness, stomach pain, muscle pain, headache, and/or fever. In these embodiments, the uses of the compositions of the invention are therapeutic and/or prophylactic uses.

In alternative embodiments, the animal is an animal which is not susceptible to clinical manifestations of the Campylobacter infection. Indeed, many non-human animals carry Campylobacter as intestinal commensals (i.e. non-pathogenic bacteria in the gastrointestinal tract). For these animals Campylobacter, and in particular Campylobacter jejuni is a non-pathogenic bacterium. In these embodiments, the uses of the compositions of the invention are non-therapeutic for the animals to which they are administered, but relate to ensuring food or feed safety for other animals (to which these animals are provided as food or feed) or to avoid contamination or reduce risk of contamination for other animals (e.g. where these other animals are in contact with the animals for which Campylobacter is a non-pathogenic bacterium, such as the case for pets or small farm animals) by disinfecting the animal and reducing the number of Campylobacter present in the gastrointestinal tract of said animal. In these embodiments, the use can be e.g. as a feed-additive, more particularly as a drinking water feed-additive, as will be detailed herein. Examples of animals for which Campylobacter jejuni is a non- pathogenic bacterium include but are not limited to avian species such poultry (e.g.

chickens, turkeys, ducks, ostriches and geese).

In particular embodiments, the animal is a non-human animal, preferably a non- human animal selected from the list consisting of cattle, sheep, pigs, goats, deer, fish, shellfish and poultry. In more particular embodiments, the non-human animal is selected from the list consisting of chicken, goose, turkey and duck. In even more particular embodiments, the non-human animal is a chicken. The terms “gastrointestinal (Gl) tract” or “gut” as used herein refers to an organ system within animals which takes in food, digests the food to extract and absorb energy and nutrients, and expels the remaining waste as feces. Organs of the gastrointestinal tract include the esophagus, stomach, first, second and third part of the duodenum, jejunum, ileum, the ileo-cecal complex, large intestine, sigmoid colon and rectum.

Campylobacter are gram negative, comma or s-shaped bacteria, which are motile via unipolar or bipolar flagella.

Campylobacter species usually live in the gut of animals, in particular in the gut of chickens.

At least a dozen species of Campylobacter have been implicated in human disease, with Campylobacter Jejuni (C.

Jejuni), Campylobacter fetus or Campylobacter Coli (C.

Coli), C.

Jejuni being the most common.

In particular embodiments of the uses or methods as taught herein, the Campylobacter is Campylobacter Jejuni, Campylobacter Coli or Campylobacter fetus.

In particular embodiments, the Campylobacter is a Campylobacter which is pathogenic to the animal.

In alternative embodiments, the Campylobacter is a Campylobacter which is not pathogenic to the animal to which the iron chelate is administered, but is pathogenic to other animals which may be contacted with products of said first animal as food or feed or in any other way.

The compositions of the present invention are envisaged to be suitable for use in different aspects of Campylobacter infections.

Reduction of number of Campylobacter The compositions of the present invention ensure a reduction of the invasion and/or transcellular migration through the gastrointestinal cells of an animal by Campylobacter by iron chelate, leads to a reduction of the total number of viable Campylobacter present in the gastrointestinal tract of an animal.

In view hereof, also provided herein is the use of iron chelate for reducing the number of Campylobacter present in the gastrointestinal tract of an animal comprising administering the iron chelate, preferably iron(lll) EDTA, in an effective amount to said animal to reduce the number of Campylobacter present in the gastrointestinal tract of said animal.

Also provided herein is a method for reducing the number of Campylobacter present in the gastrointestinal tract of an animal, the method comprising the step of administering iron chelate, preferably iron(lll) EDTA, in an effective amount to said animal to reduce the number of Campylobacter present in the gastrointestinal tract of said animal.

The number of (viable) Campylobacter in the gastrointestinal tract of an animal may be reduced by the uses and methods as taught herein.

The number of Campylobacter in the gastrointestinal tract of an animal may be determined by any known method for determining the number of (viable) bacteria in the gastrointestinal tract of an animal.

For example, the number of (viable) Campylobacter may be determined based on the number of colony-forming units (cfu). The term “colony forming unit” or “cfu” refers to a unit used to estimate the number of cells present in a sample of the animal, such as a feces sample, based on their ability to give rise to colonies under specific conditions of nutrient medium, temperature and time. Theoretically, one viable cell can give rise to one colony through replication.

1000 cfu of Campylobacter are typically sufficient to infect a human and cause disease in a human. Therefore, in particular embodiments, the effective amount of iron chelate as taught herein is an amount of iron chelate sufficient to reduce the number of Campylobacter (or cfu of Campylobacter) in the gastrointestinal tract of an animal to a number (or cfu of Campylobacter) that is unlikely to cause infections in humans, preferably to at most 900 cfu, at most 800 cfu, at most 700 cfu, at most 600 cfu, at most 500 cfu, at most 400 cfu, at most 300 cfu, at most 200 cfu, at most 100 cfu, at most 50 cfu, at most 25 cfu, at most 10 cfu, at most 5 cfu, preferably at most 100 cfu. The effective amount may depend on the type of iron chelate used and/or the size of the animal.

In particular embodiments, the uses and methods for reducing the numbers of Campylobacter in the gastrointestinal tract of an animal as taught herein include the reduction of the number of Campylobacter in the gastrointestinal tract of the animal by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or at least 99%, preferably at least 50%, more preferably at least 90%. In particular embodiments, the number of Campylobacter in the gastrointestinal tract of the animal treated with the iron chelate as taught herein, is reduced to a number of Campylobacter undetectable by methods known in the art. In particular embodiments, Campylobacter may be substantially eradicated from the gastrointestinal tract of the animal treated by the uses or method as taught herein.

In particular embodiments, the uses and methods for reducing the numbers of Campylobacter as taught herein include the reduction of the number of cfu of Campylobacter by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or at least 99%, preferably at least 50%, more preferably at least 90%.

The number of cfu of Campylobacter that would be ingested by a human if they ate meat from anon-animal infected by Campylobacter may be related to the number of Campylobacter in the gastrointestinal tract of the infected non-animal at the time of slaughter of said non-animal but also represents on other factors such as the amount of contamination of the meat with the contents of the gastrointestinal tract of the non- human animal at the time of slaughter. Accordingly, the uses and methods as taught herein are preferably combined with methods for slaughtering non-animals animals, which aim at a reduced contamination of the meat by Campylobacter.

Reducing the colonization The compositions of the present invention comprising iron chelate have been found to make Campylobacter less virulent and/or less capable of infecting animals. This may even occur if the total number of Campylobacter in the gastrointestinal tract does not decrease. Furthermore, the iron chelates are envisaged to affect the metabolism of Campylobacter and make them less adaptive to the environment so that they cannot colonize the gastrointestinal tract of an animal and are less likely to be transmitted to other animals.

In view hereof, further provided herein is the use of iron chelate, such as iron(lll) EDTA, for preventing or reducing the colonization of the gastrointestinal tract of ananimal with Campylobacter, comprising administering iron chelate in an effective amount to said animal to prevent or reduce the colonization of the gastrointestinal tract of said animal .Also provided herein is iron chelate for use in a method of preventing or reducing the colonization of the gastrointestinal tract of an animal with Campylobacter.

Likewise, further provided herein is a method for preventing or reducing the colonization of the gastrointestinal tract of an animal, the method comprising the step of administering iron chelate, preferably iron(lll) EDTA, in an effective amount to said animal to prevent or reduce the colonization of the gastrointestinal tract of said animal.

Campylobacter have the ability to form biofilms which enables the Campylobacter to survive in environments it normally would not be able to. The Campylobacter present in the biofilm are able to share nutrients and are sheltered from harmful factors in the environment. A biofilm comprises any group of microorganisms in which cells adhere to each other and often also to a surface, such as the walls of the gastrointestinal tract.

It is noted that by reduction of the number of Campylobacter in the intestinal tract and reducing their ability to colonize the intestinal tract of an animal, the Campylobacter will not proliferate and thus will not be able to create/continue a biofilm. Thus, the methods of the present invention also prevent and reduce biofilm formation by Campylobacter.

Reducing transmission By reducing the number of Campylobacter in the intestinal system, the transmission thereof to other animals is also reduced. Accordingly, a further aspect provides the use of iron chelate for the prevention or reduction of transmission of Campylobacter infection from one non-human animal to another, comprising administering said iron chelate, to said animals.

Also provided herein is a method for preventing or reducing transmission of Campylobacter infection from one animal to another, the method comprising a step of administering iron chelate, preferably iron{lll) EDTA, to said animals.

Also provided herein is iron chelate for preventing or reducing spread of Campylobacter infection within a flock or herd of animals. For example, iron chelate, preferably iron(lll) EDTA, may be used for preventing or reducing spread of Campylobacter infection within a flock of chickens.

Disinfection The compositions of the invention generally allow the disinfection of an animal for consumption, more particularly prior to its slaughter for human consumption. In particular embodiments, the animal is a non-human animal and the iron chelate, is administered to said animal prior to slaughter of said animal.

In particular embodiments, the uses or the methods as taught herein comprise a step of slaughtering the non-human animal, preferably after the administration of iron chelate to said animal.

Further provided herein is the use of iron chelate, preferably iron(lll) EDTA, for reducing the amount of Campylobacter in meat comprising providing a non-animal with iron chelate and subsequently preparing a meat product from the non-human animal.

Also provided herein is a method for reducing the amount of Campylobacter in meat comprising the steps of: providing a non-human animal with iron chelate, preferably iron(lll) EDTA, and preparing a meat product from the animal.

It might be beneficial to reduce the presence of Campylobacter on food, feed or food or feed product intended for human consumption, thereby reducing the risk of infection of humans with Campylobacter.

Accordingly, also provided herein is a method of disinfecting a food, feed or food or feed-product comprising a step of administering iron chelate, preferably iron(lll) chelate, in an effective amount to the food, feed or food- or feed product to reduce the amount of Campylobacter in the food, feed or food or feed product.

Likewise, also provided herein is the use of iron chelate, preferably iron(II} chelate, for disinfecting a food, feed or food- or feed-product by administering an effective mount of iron chelate, preferably iron(lll) chelate, to the food, feed or food or feed product to reduce the amount of Campylobacter in the food, feed or food or feed product.

In particular embodiments, the food or food product is for human consumption. Non- limiting examples of food and food products are meat products (e.g. fresh meat products, processed meat products, chilled meat products, frozen meat products, cooked meat products), eggs and raw (unpasteurized) milk products. In particular embodiments, the food or food product is a poultry meat product, preferably a chicken meat product.

Treatment of pathogenic infection Although unlike humans, most non-human animals carry Campylobacter as intestinal commensals (i.e. non-pathogenic bacteria in the gastrointestinal tract), Campylobacter may also contribute to gastrointestinal disease or cause other disorders. For instance, in young non-human animals, such as weaning piglets, but also in humans, Campylobacter may cause gastrointestinal disease.

Clinical symptoms of a Campylobacter infection include diarrhea (which can be bloody), fever, and abdominal cramps. The diarrhea may be accompanied by nausea and vomiting. These symptoms usually start within 2 to 5 days after exposure and last about a week.

As described elsewhere herein, iron chelate prevents and/or reduces invasion of and/or transcellular migration through the epithelial cells in the gastrointestinal tract by Campylobacter. In view hereof, it is beneficial to administrate iron chelate to animals (both human and non-human animals) to treat or prevent a gastro-intestinal infection with Campylobacter, which is also known as campylobacteriosos. Typically, in these embodiments, the Campylobacter is C. jejuni or C. coli, though C. fetus may also cause diarrheal illnesses.

Campylobacter upsaliensis is typically found in young dogs and cats.

Campylobacter species, including C. jejuni and C. fetus, have also been associated with a number of extra-intestinal illnesses. For instance campylobacter infections have been associated with Guillain-Barre Syndrome (GBS), post-infectious irritable bowel syndrome (IBS), and reactive arthritis.

Campylobacter fetus has also been associated with abortion in sheep and cattle.

In particular embodiments, the animal, such as the human, is an immunocompromised animal, which is more susceptible to disease caused by Campylobacter. More particularly, immunocompromised animals are susceptible to systemic infections caused by Campylobacter. For instance, in animals with weakened immune systems, such as suffering from a blood disorder such as thalassemia and hypogammaglobulinemia, AIDS, or receiving chemotherapy, Campylobacter can spread to the bloodstream and cause a life-threatening infection.

Accordingly, further provided herein is use of iron chelate, preferably iron(lll) EDTA, in the treatment of a bacterial infection by Campylobacter (or Campylobacter infection) in an animal as detailed above.

Likewise, also provided herein is a method of treating a Campylobacter infection in an animal, comprising administering to the animal an effective amount of iron chelate to treat a Campylobacter infection.

The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed bacterial infection of the gastrointestinal tract, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of a bacterial infection of the gastrointestinal tract.

Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms, diminishment of extent of disease, and the like.

The term “bacterial infection” as used herein, refers to the invasion of an animal (“host”) body tissue by a pathogenic or disease causing bacteria, their multiplication, and the reaction of subject's body tissues to the infectious bacteria and the toxins they produce.

Pathogenic bacteria are usually distinct from the usually harmless bacteria of the normal intestinal flora.

The bacteria can cause direct damage to the cell by attaching to and/or invading into the host cells, using the host cell for nutrients and producing waste products and/or toxins.

Alternatively, the bacteria can cause indirect damage by provoking an excessive or inappropriate immune response that inadvertently damages host cells.

Bacterial infections of the gastrointestinal tract may be determined methods known by the skilled person for identifying bacterial infections of the gastrointestinal tract.

For example, studying the appearance (e.g. colour and consistency) of the animal's faeces, and/or demonstrating the presence of the pathogenic bacteria in the GI tract orin a fecal sample of the affected animal by special culture techniques and molecular technologies (e.g.

ELISA, PCR), depending on the suspected infectious bacteria.

In particular embodiments, the uses or methods as taught herein may comprise identifying the bacterial infection prior to starting the treatment.

In particular embodiments the efficacy of the treatment is followed up by determining the presence of clinical symptoms of the disease (e.g. diarrhoea, weight loss etc.).

The term “diarrhoea”, as used herein, refers to a condition of having three or more loose or liquid stools or bowel movements per day. In particular embodiments, the diagnosis of diarrhea is performed by visual inspection of the feces (in accordance with a pre-determined scoring system).

Additionally or alternatively, the efficacy of the treatment is followed up by determining the presence of Campylobacter in the feces. In particular embodiments, the duration of the treatment is dependent on whether or not Campylobacter are still excreted by the animal under treatment and/or whether or not the clinical symptoms are reduced or eliminated. Accordingly, in particular embodiments, the methods comprise determining at least once, preferably at regular intervals after the start of treatment, whether or not Campylobacter are still present in the feces of the animal being treated. In particular embodiments, the treatment is stopped once it is determined that the feces of the human subject is substantially free of pathogenic bacteria.

The quantity (or effective amount) of the iron chelate, preferably iron(lll) EDTA, administered to the animal to be treated can be adjusted in accordance with the purpose to be attained, the nature of the subject, the condition under consideration, as well as the type of formulation wherein the iron chelate is comprised, and any other relevant facts that may modify the activity of iron chelate or the response of the animal, as is well known by those skilled in the art. The quantity of iron chelate to be used for prevention is usually lower than those to be used for the reduction or elimination of an already established bacterial infection.

The inventors found through in vivo experiments that administering an iron chelate to an animal, e.g. in the drinking water of said animal is effective in preventing or reducing the prevalence of Campylobacter in the gastrointestinal tract of said animal.

Different dosages have been tested. In particular embodiments, a dosage of between 5 and 100 ppm in the drinking water is envisaged. In further embodiments, the dosage is between 10-50 ppm, such as between 15 and 40 ppm, such as between 18, 5 and 37 ppm. In alternative embodiments, a dosage of 5 to 20 mg per kilogram of body weight of said animal, or administration of between 20-150 mg/liter drinking water. In particular embodiments, a stock solution is used, which is then diluted in the drinking water. For instance, 50 ppm of a stock solution containing 37% iron{lINEDTA will result in a concentration of 18.5 ppm iron(lll) EDTA in the drinking water.

In particular embodiments this dosage is effective in preventing the invasion and/or transcellular migration through the gastrointestinal cells of the animal by Campylobacter or reducing the number of Campylobacter invading and/or transmigrating through to the gastrointestinal cells of the animal, reducing the number of Campylobacter present in the gastrointestinal tract of an animal, preventing or reducing the colonization of the gastrointestinal tract of an animal with Campylobacter, preventing or reducing transmission of Campylobacter infection from one non-human animal to another and/or treating a Campylobacter infection, while not being toxic for the environment.

As animals, preferably non-human animals, typically consume daily a larger volume of drinking water compared to feed, the dosages of iron chelate, such as iron{lll) EDTA, per liter of drinking water are typically lower than the dosages of iron chelate per kilogram of feed required to achieve a similar total daily intake of the iron chelate, such as iron{lll) EDTA.

Accordingly, the administration by way of drinking water is preferred.

In particular embodiments, the iron chelate is administered to said animal at a daily dosage from 10 to 1500 mg, from 10 to 600 mg, from 10 to 300 mg, from 50 to 250 mg, from 75 to 225 mg, preferably from 90 to 200 mg per liter of drinking water of said animal, preferably wherein said animal is a non-human animal.

The dosages will depend to some extent on the animal.

In particular embodiments, iron chelate, such as iron(lll) EDTA, is administered to said animal at a daily dosage from 1.5 to 25 mg, from 2 to 25 mg, from 3 to 25 mg, from 4 to 25 mg, from 5 to 25 mg per kilogram of body weight of said animal.

In particular embodiments, such as for chickens with an average weight of about 2kg, the daily dosage ranges from 0.5 to 25 mg, from 1 to 25 mg, from 2 to 25 mg, from 2 to 10 mg, from 2 to 8 mg, from 3 to 6 mg per kilogram of body weight of said animal.

The iron chelate, such as but not limited to iron(lll) EDTA, may be administered to the animal once daily, multiple times daily, for example two, three, or four times per day, or continuous during a certain time period, for example, by addition of the iron chelate in the drinking water, food or feed of the animal.

In certain embodiments, ad libitum administration is envisaged during a period of at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or at least two weeks.

In certain embodiments, the iron chelate is provided to the animal in their drinking water or feed, which the animal may drink or eat ad libitum.

The timing of administration of iron chelate to the animal may be determined by the circumstances and the envisaged effect.

For instance, in particular embodiments as described herein, the iron chelate is used to prevent infection and/or reduced prevalence of Campylobacter in the animal, in particular where reduced presence or absence of Campylobacter in the animal at given moment is important or where the animal is expected, as a result of circumstances or events, to become more susceptible to infection. In these embodiments it can be of interest to administer the iron chelate timely before that moment or those circumstances/events. For instance, in particular embodiments, the iron chelate is administered to the non-human animal at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least days, or at least two weeks before slaughter. Chickens are often colonized by Campylobacter between 7 and 8 days before slaughter. Accordingly, in particular embodiments, the iron chelate is administered to the non-human animal less than 10 days, less than 9 days, less than 8 days or less than 7 days before slaughter of the non-human animal. This may allow the disinfection of the non-human animal and the 10 reduction of colonization of the gastrointestinal tract of the non-human animal before slaughter. In particular embodiments, the iron chelate is administered to the non- human animal at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or at least two weeks before transportation of said animals, e.g. to prevent cross-infection. In particular embodiments, the iron chelate is administered to the animal at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or at least two weeks before an event that can negatively affect the health of said animals and makes them susceptible to infection, such as a vaccination, change in feed, transition period for dairy animals such as cows, etc.

The skilled person will understand that if the iron chelate is administered to the animal in order to prevent the invasion and/or transcellular migration through the gastrointestinal cells of the animal by Campylobacter, to reduce the number of Campylobacter invading and/or transmigrating through the gastrointestinal cells of the animal, to reduce the number of Campylobacter present in the gastrointestinal tract of an animal, to prevent or reduce the colonization of the gastrointestinal tract of an animal with Campylobacter, or to prevent or reduce transmission of Campylobacter infection from one non-human animal to another, the iron chelate is preferably administered to the animal in the period before such an event typically occurs in said animal.

In particular embodiments, the as described herein, the iron chelate is used to cure infection of an animal by Campylobacter, whereby timing may be dictated by the occurrence of symptoms of said disease. Accordingly, in particular embodiments, where the envisaged use is for treatment of infection, the iron chelate may be administered to said animal daily at least for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or for at least two weeks from the start of treatment.

In particular embodiments, the efficacy and duration of the uses and methods as taught herein is followed up by determining the presence and/or the number of Campylobacter in the faeces. In particular embodiments, the duration of the use or administration of the iron chelate, is dependent on whether or not Campylobacter are still excreted by the animal. Accordingly, in particular embodiments, the uses and methods as taught herein comprise determining at least once, preferably at regular intervals after the start of administration of the iron chelate whether or not Campylobacter are still present in the feces of the animal to which the iron chelate is administered. In particular embodiments, the administration is stopped once it is determined that the feces of the animal is substantially free of Campylobacter.

In alternative embodiments, where the use is therapeutic, the efficacy and duration of the uses and methods as taught herein is determined by the presence or absence of symptoms of said infection.

In particular embodiments, the iron chelate, is administered orally. Oral administration ensures that the iron chelate reaches the gastrointestinal tract of the animal and/or allows easy and less labor-intensive administration to non-human animals.

The recitation “administered orally”, “oral administration”, “oral delivery”, “oral consumption”, or “administered per os” as used herein, refers to a route of administration to an animal where a substance or composition is taken through the mouth of said animal. One or more substances taken through the mouth may have a systemic or local (e.g. in the mount, oesophagus, stomach, intestines and/or rectum) effect in the animal. The iron(lll) EDTA may be ingested orally in the form of a powder, a tablet, a capsule, a pastille or a liquid preparation. If the iron(lll) EDTA is in powder form, it may be solubilized in any type of liquid, for example in water, before administration to the animal.

In certain embodiments, the iron(lll) EDTA may be configured for oral administration The recitation “configured for oral administration” as used herein refers to the capability of the the iron(lll) EDTA to be administered through the oral cavity of an animal to the digestive system. Oral delivery of the iron(lll) EDTA allows it to enter the gastrointestinal tract of the subject.

In certain embodiments, the iron{lll) EDTA may be in the form of a powder, a tablet, a capsule, a pastille, or a liquid preparation, preferably a powder. In particular embodiments, the iron chelate used the uses or methods as described herein, is in the form of an animal feed, animal drinking water, animal drinking water supplement, feed ingredient or feed supplement that comprises the iron chelate, preferably iron(lll) EDTA, preferably wherein the animal is a non-human animal.

As used herein, the term “feed” encompasses food for non-human animal consumption. Examples of suitable feed materials include but are not limited to brans, such as wheat bran, rice bran, barley bran, and millet bran; food processing by- products, such as soybean-curd residue, starch pulp, copra meal, sake cake, soy sauce cake, brewer's grains, sweet potato distiller's residue, and juice pulp of fruits and vegetables; cereals, such as corn, rice, wheat, barley, and oat; oil seed meals, such as soybean meal, rapeseed meal, cotton seed meal, linseed meal, sesame meal and sunflower meal; animal origin feeds, such as fish meal, casein, dried skim milk, dried whey, meat and bone meal, meat meal, feather meal, and blood meal; leaf meals, such as alfalfa meal; and the like.

The iron{lll} EDTA can be readily administered by mixing it directly into animal feed, or separately from the feed as a supplement in an edible carrier to be later mixed with the feed.

In particular embodiments, the iron chelate, preferably iron(lll) EDTA, is added to the animal feed or fodder or to the animal drinking water in an effective amount, preferably wherein the animal is a non-human animal.

Also provided herein is feed or fodder or drinking water comprising iron chelate. The iron chelate, preferably iron(lll) EDTA, may be present in a premix in a concentration that is determined according to its intended mixing ratio with the animal feed or animal drinking water; the iron chelate content of a premix may be e.g. the 10-100 fold of the values envisaged for feed or fodder or animal drinking water.

In particular embodiments, the feed and/or drinking water additive comprises at least 25% ((w/w) or (w/v)), at least 30% ((w/w) or {w/v)), at least 35% ((w/w) or (w/v)), or at least 40% ((w/w) or {w/v)) of an iron chelate, more preferably iron(lll} EDTA. For example, the additive may comprise about 37% (w/v) iron(lll} EDTA. In particular embodiments, the iron chelate is dissolved in water and the composition further comprises colorants and preservatives in standard amounts.

In particular embodiments, the feed and/or drinking water additive as described herein comprises one or more iron chelates as the only active ingredient. In particular embodiments, the feed and/or drinking water additive comprises only one iron chelate, preferably iron(lll) EDTA, as the only metal chelate present in the composition. In further particular embodiments, the feed and/or drinking water additive comprises iron chelate, preferably iron(lll) EDTA, as the only active ingredient.

If administered separately from the animal feed or fodder or the animal drinking water, dosage forms of the iron chelate as described herein can be prepared by combining it with non-toxic acceptable edible carriers to make either immediate release or slow release formulations, as is well known in the art. Such edible carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol {and used as a supplement or top dressing on feed). The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeutically valuable substances.

Further provided herein is the use of iron chelate, preferably iron(lll) EDTA, or compositions comprising iron chelate, preferably iron(lll) EDTA, as a feed ingredient, feed supplement or animal drinking water supplement.

As used herein, the term “feed/drinking water supplement’ refers to an ingredient, additive, component or supplement suitable for incorporation in animal feed and/or drinking water.

In particular, iron chelate, is envisaged as a feed ingredient, feed supplement and/or animal drinking water supplement for non-human animals bred for human consumption carrying a large amount of Campylobacter in their gastrointestinal tract, such as poultry, thereby being a potential source of human infection.

In certain embodiments, one or more pharmaceutical excipients may be added to the iron chelate, preferably iron(lll) EDTA. Suitable pharmaceutical excipients depend on the dosage form and identities of the active ingredients and can be selected by the skilled person. As used herein, “carrier” or “excipient” includes any and all solvents, diluents, buffers (such as neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as EDTA or glutathione), amino acids (such as glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilisers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. For example, the one or more pharmaceutical excipients may be selected from carboxymethyl cellulose, silicon dioxide, propylene glycol, polyethylene glycol and oil (e.g. peanut oil, olive oil, sesame oil, corn oil), sodium carbonate, whey powder, corn starch, lactose, sucrose and soy flakes. The carriers may allow either immediate release or slow release of the active ingredient. The use of such media and agents for pharmaceutical active substances is well known in the art. Such materials should be non-toxic and should not interfere with the activity of the active ingredients.

herein is a pharmaceutical composition comprising iron chelate, preferably iron(lll) EDTA.

The skilled person will understand that the use of a pharmaceutical composition comprising iron chelate in the uses and methods as taught herein, is also provided herein. For example, an aspect provides a pharmaceutical composition comprising iron chelate, preferably iron(lll) EDTA for use in the treatment of Campylobacter infections of the gastrointestinal tract in an animal, preferably a human.

Typical pharmaceutical dosage forms for internal administration are orally administrable dosage forms, such as pastes, solutions, tablets, etc.. However, injectable compositions are also envisaged. The compositions as taught herein may also be medicated fodders, feeds, nutriments, premixes, drinking waters and drinking water additives.

The concentration of the iron chelate in pharmaceutical compositions as taught herein depends on different factors, among others on the purpose to be attained (prevention or therapy), on the severity of the already established disease and on the type of the composition concerned.

In particular embodiments, the pharmaceutical composition comprises iron chelate, preferably iron(lll) EDTA, and one or more pharmaceutical excipients as described elsewhere herein.

In particular embodiments, the pharmaceutical composition as described herein comprises one or more iron chelates, as the only active ingredient present in the composition. In further particular embodiments, the pharmaceutical composition comprises iron chelate, preferably iron(II) EDTA, as the only active ingredient.

The terms “active ingredient” or “active component” can be used interchangeably and broadly refer to a compound ar substance which, when provided in an effective amount, achieves a desired therapeutic and/or prophylactic outcome(s). Typically, an active ingredient may achieve such outcome(s) through interacting with and/or modulating living cells or organisms. The term “active” in the recitations “active ingredient” or “active component” refers to “pharmacologically active” and/or “physically active”.

metal chelates have been shown by present inventors to reduce the number of Campylobacter in the gastrointestinal tract, by preventing or reducing the invasion and/or transcellular migration through the gastrointestinal cells of the animal by Campylobacter, thereby preventing or reducing the colonization of the gastrointestinal tract with Campylobacter, without requiring the addition of antibiotics. While antibiotics aim to kill the Campylobacter, the metal chelates of the invention flush out the Campylobacter. Contrary to antibiotics however, the use of the metal chelates herein will not induce resistance. Thus, the metal chelate of the present invention can be used as an alternative to antibiotics as feed ingredient, feed supplement, animal drinking water supplement and/or as therapeutic agent. Alternatively, the iron chelate, more particularly iron (Ill) EDTA, can also be of use in combination with antibiotics, such that concentrations of antibiotics can be reduced. Furthermore, it will also be clear for the skilled person that the pharmaceutical composition or a feed comprising a metal chelate could also be employed for the uses as described herein. The following examples are meant to illustrate the present invention and should not be construed as a limitation of its scope.

EXAMPLES Example 1. Iron(lll} chelate is effective in preventing Campylobacter from infiltrating the human intestinal cells in vitro The aim of this study was to investigate the effect of iron(lll) EDTA on adhesion and invasion ability of Campylobacter strains to intestinal cells. Materials and methods In order to investigate the in vitro efficacy of chelated iron, the formulations shown in Table 1 were prepared, which were evaluated at a concentration of 150 ppm on the attachment and internalization of Campylobacter to human model cells. The chelates used were Zinc disodium EDTA and iron potassium EDTA.

Table 1. Formulations for in vitro testing Iron chelate | Zinc chelate [een wn a [mw 9 B jee

ET C. jejuni 33291 (study 1) and field strains (study 2) were isolated from chicken caecae and grown at 42 °C for 48 hours under microaerophilic conditions on Columbia blood agar plates, and then mixed with one of the formulations of Table 1.

Human carcinoma epithelial cells (HELA, Hep) or human colon cells (Caco-2) were cultured in 24 well tissue culture trays (1 x 10° cells per well) in DMEM culture medium supplemented with 10% bovine serum and 10 pg/ml penicillin and 10 pg/ml streptomycin under low CO: (5%) at 37 °C until a monolayer formed (72 hours).

The culture medium was removed from the monolayer cells, the monolayer cells were washed with PBS and incubated in one of the formulations of Table 1 (150 ppm in culture medium). Adhesion assay The monolayer of human cells was infected with 6 '®log CFU/ml of the C. jejuni 33291 strain and incubated at 41.5 °C for 3 hours to allow adhesion of the bacteria to the human cells. Unbound bacteria were washed 3x in PBS, and adherent bacteria were enumerated in maximum recovery diluent, plated on Columbia Blood Agar and modified CCDA, and incubated micro-aerobically for 48 hours at 41.5 °C. Invasion assay The monolayer of human cells was infected with 6 ®log CFU/ml of the C. jejuni 33291 strain and incubated at 41.5 °C for 3 hours to allow adhesion of the bacteria to the human cells. Unbound bacteria were washed 3x in PBS. To estimate invasion levels, gentamycin {100 pg/ml) was added for 30 minutes to lyse extracellular bacteria. The monolayers of human cells were then lysed with ice cold water and the intracellular bacteria were enumerated as described above. Results The formulations A-E did not significantly affect the attachment C. jejuni (strain 33291) tothe human intestinal cells (Figure 1, table 2). However, a significant inhibition of cell invasion by C. jejuni was observed for formulations A (100% iron chelate), B C (80% iron chelate) and C (50% iron chelate) (Figure 1, table 2 and Figure 2, Table 3), especially for C. jejuni 33291 (Figure 1, table 2).

Example 2. Iron(lll} chelate is effective in inhibiting the internalization of Campylobacter jejuni into the intestinal cells in vivo Pre-clinical trial On a commercial farm regularly experiencing Campylobacter infections, the results of 4 broiler houses were compared with each other.

Two houses were treated from week 4 until week 6 (slaughter) with 50 ppm chelated iron, the other 2 houses served as controls.

Thinning was performed at week 5 (= 35 days). From day 14, the houses were tested for Campylobacter every 7 days and at slaughter 10 individual birds per house were tested for Campylobacter infection.

All tests were negative for all 4 houses.

The average slaughter weight indicates that the product did not have a negative effect on bird performance (Figure 3, Table 4). Clinical trial Farms were swabbed (e.g. swabbing of the drinker and feeders, and cloacal swabs) pre-thin to identify if Campylobacter was present using PCR analysis in conjunction with MP13D detection method (Based on ISO 10272-1:2017 - Horizontal Method for detection and enumeration of Campylobacter spp. - Part 1: Detection mothod). If negative, the birds received a37% solution of chelated iron in water continuously through the drinking water for the duration of 2 weeks prior to slaughter.

The animals were tested by caecal enumeration (culturing of caecal tonsil samples on selective agar plates and counting the number of colonies) from thin until slaughter.

When a flock is becoming Campylobacter positive, all birds typically become positive very quickly.

This was not observed in these product treated flocks (Figure 4). Conclusion The in vivo studies demonstrated that the product is not negatively impacting bird performance (Figure 3) and preliminary results confirm the reduction of Campylobacter infection pressure in broiler flocks upon treatment with chelated iron (Figure 4). The invention is also characterized by the following statements:

1. Methods are provided for preventing or reducing the prevalence of Campylobacter in a non-human animal host, said method comprising administering iron(lll) chelate to said animal.

2. In particular embodiments of said methods said iron (Ill) chelate is iron(lll) EDTA, DTPA, HEDTA, MGDA or GLDA.

3. In particular embodiments of said methods said animal is a live-stock animal and wherein said iron(lll) chelate is administered to said animal prior to slaughter of said non-human animal.

4. In particular embodiments of said methods said animal is a live-stock and said wherein said iron(lll) chelate is administered to said animal prior to envisaged events which increase the risk of infection or cross-infection of said animal.

5. In particular embodiments of said methods the iron(lll) chelate is administered in the animal drinking water.

6. In particular embodiments of said methods said animal is provided with drinking water comprising at least Sppm iron(lll) chelate.

7. In particular embodiments of said methods said animal is allowed access to the drinking water ad libidum.

8. In particular embodiments of said methods the Campylobacter is Campylobacter Jejuni or Campylobacter Coli.

9. Also provided is Iron(lll) EDTA for use in the treatment of a Campylobacter infection in an animal suffering from symptoms of said infection.

10. Also provided is the use of an iron(lll} chelate for preventing the invasion of gastrointestinal cells of an animal by Campylobacter or reducing the number of Campylobacter invading the gastrointestinal cells of an animal, wherein said use is not a method of treatment of the animal or human body.

11. Also provided are methods for reducing the susceptibility of a live-stock animal to infection by Campylobacter, said method comprising, administering iron(lll) chelate to said animal.

12. Also provided is the use of iron(lll) EDTA for preventing the invasion of gastrointestinal cells of an animal by Campylobacter or reducing the number of Campylobacter invading the gastrointestinal cells of an animal comprising administering iron(lll} EDTA in an effective amount to said animal to prevent the invasion of gastrointestinal cells of an animal by Campylobacter or to reduce the number of Campylobacter invading the gastrointestinal cells of the animal.

13. Also provided is the use of iron(lll) EDTA for preventing or reducing the transcellular migration of Campylobacter through the gastrointestinal cells of an animal comprising administering iron({lll) EDTA in an effective amount to said animal to prevent or reduce the transcellular migration of Campylobacter through the gastrointestinal cells of the animal.

14. Also provided is the use of iron(lll) EDTA for reducing the number of Campylobacter present in the gastrointestinal tract of an animal comprising administering iron(lll) EDTA in an effective amount to said animal to reduce the number of Campylobacter present in the gastrointestinal tract of said animal.

15. Also provided is the use of iron(lll) EDTA for preventing or reducing the colonization of the gastrointestinal tract of an animal with Campylobacter comprising administering iron(lll) EDTA in an effective amount to said animal to prevent or reduce the colonization of the gastrointestinal tract of said animal.

16. Also provided is the use of iron(lll) EDTA for the prevention or reduction of transmission of Campylobacter infection from one animal to another comprising the administration of the iron(lll) EDTA to said animals.

17. In particular embodiments of said uses said animal is a non-human animal and wherein said iron{lll) EDTA in administered to said animal prior to slaughter of said non-human animal.

18. In particular embodiments of said uses the iron(lll) EDTA is in the form of an animal feed, animal drinking water, feed ingredient, feed supplement or animal drinking water supplement that comprises the iron(lll) EDTA.

19. Also provided is Iron(lll}) EDTA for use in the treatment of a Campylobacter infection in an animal, comprising administering an effective amount of said iron(IlEDTA to said human subject to treat said Campylobacter infection, preferably wherein said animal is a human.

20. In particular embodiments of said uses the iron(lll}) EDTA is administered to said animal orally.

21. In particular embodiments of said uses the iron(lll} EDTA for use according to any one of claims 8 to 10, wherein the iron(lll) EDTA is administered once a day or the animal is allowed to access said composition ad libidum.

22. In particular embodiments of said uses the Campylobacter is Campylobacter Jejuni or Campylobacter Coli.

23. In particular embodiments, the iron(lll) EDTA is comprised in a pharmaceutical composition or an animal feed.

Claims (12)

CONCLUSIESCONCLUSIONS 1. Een methode voor het voorkomen of beperken van het voorkomen van Campylobacter in een dierlijke gastheer, waarbij de methode er in bestaat om ijzer(lll) chelaat toe te dienen aan de dierlijke gastheer.A method of preventing or limiting the occurrence of Campylobacter in an animal host, the method comprising administering iron (III) chelate to the animal host. 2. De methode volgens conclusie 1, gekenmerkt door het feit dat de methode de invasie van gastro-intestinale cellen van het dier door Campylobacter voorkomt en/of het aantal Campylobacter dat de gastro-intestinale cellen van het dier binnendringt vermindertThe method according to claim 1, characterized in that the method prevents the invasion of gastrointestinal cells of the animal by Campylobacter and / or reduces the number of Campylobacter entering the gastrointestinal cells of the animal. 3. De methode volgens conclusie 1 of 2, dat erdoor gekenmerkt is dat het ijzer (Ill) chelaat ijzer (ll!) EDTA, DTPA, HEDTA, MGDA or GLDA is.The method according to claim 1 or 2, characterized in that the iron (III) chelate is iron (II!) EDTA, DTPA, HEDTA, MGDA or GLDA. 4. De methode volgens een van de voorgaande conclusies waarbij het dier vee is en het ijzer (Ill) chelaat wordt toegediend voor het slachten van het dier.The method of any preceding claim wherein the animal is livestock and the iron (III) chelate is administered before slaughtering the animal. 5. De methode volgens een van conclusies 1 tot 3, dat erdoor gekenmerkt is dat het dier vee is en het ijzer (lll) chelaat wordt toegediend voor voorziene omstandigheden die het risico op infectie of kruis-infectie verhogen.The method according to any one of claims 1 to 3, characterized in that the animal is livestock and the iron (III) chelate is administered for anticipated conditions that increase the risk of infection or cross-infection. 6. De methode volgens een der voorgaande conclusies dat erdoor gekenmerkt is dat het ijzer (lll) chelaat toegevoegd wordt aan het drinkwater van het dier.The method according to any one of the preceding claims, characterized in that the iron (III) chelate is added to the drinking water of the animal. 7. De methode volgens conclusie 6, dat erdoor gekenmerkt is dat het drinkwater ten minste 5ppm ijzer(II!) chelaat bevat.The method according to claim 6, characterized in that the drinking water contains at least 5ppm iron (II!) Chelate. 8. De methode volgens conclusie 6 of 7, dat erdoor gekenmerkt is dat het dier ad libidum toegang geeft aan het drinkwater.The method according to claim 6 or 7, characterized in that the animal provides ad libidum access to the drinking water. 9. De methode volgens een der voorgaande conclusies, dat erdoor gekenmerkt is dat Campylobacter is gekozen uit Campylobacter Jejuni of Campylobacter Coli.The method according to any one of the preceding claims, characterized in that Campylobacter is selected from Campylobacter Jejuni or Campylobacter Coli. 10. ljzer(lll) EDTA voor gebruik in de behandeling van een Campylobacter infectie in een dier dat clinische symptomen vertoont van deze infectie.10. Iron (III) EDTA for use in the treatment of a Campylobacter infection in an animal showing clinical signs of this infection. 11. Het gebruik van ijzer (lll) chelaat voor het verhinderen van de invasie van gastro-intestinale cellen van een dier door Campylobacter of voor het verminderen van het aantal Campylobacter die de gastro-intestinale cellen van het dier invaderen, waarbij het gebruik geen medische behandeling is van het menselijk of dierlijk lichaam.11. The use of iron (III) chelate to prevent the invasion of gastrointestinal cells of an animal by Campylobacter or to reduce the number of Campylobacter invading the gastrointestinal cells of the animal without the use treatment of the human or animal body. 12. Een methode voor het beperken van de gevoeligheid van een vee dier aan infectie door Campylobacter, waarbij de methode het toedienen van ijzer (lll) chelaat aan het dier.A method for limiting the susceptibility of a livestock animal to infection by Campylobacter, the method comprising administering iron (III) chelate to the animal.